After years of neglect, glial cells are finally registering on drug developers' radar. Evidence has accrued over the last seven years that glia are activated by opioids (e.g. morphine, methadone, meperidine, and oxycodone) and that this opioid-induced glial response suppresses opioid analgesia, resulting in the development of opioid tolerance and dependence. A literature has developed linking opiate efficacy and side effects to their influence on glial cells within the central nervous system (CNS) via the signaling pathway mediated by toll-like receptor 4 (TLR4). In a recent report, the Yin lab has provided the first direct evidence that morphine creates its neuroinflammatory effects by binding to the TLR4 accessory protein, MD-2, and inducing TLR4/MD-2 dimerization and subsequent TLR4 signaling activation in a similar fashion to the classical TLR4 ligand, lipopolysaccharide (LPS; endotoxin). Further, morphine induces neuroinflammation solely through its binding in a specific LPS-binding pocket of MD-2, indicating that disruption of the essential TLR4/MD-2 interactions is sufficient to suppress morphine-induced neuroinflammation. The overall objective of the current proposal is to validate the TLR4/MD-2 complex as a feasible target to suppress morphine-induced neuroinflammation by exogenous chemical probes. Our central hypothesis is that by blocking the formation of the TLR4/MD-2 complex, opioid-induced inflammatory response can also be suppressed and thereby enhance morphine analgesia. The proposed research is significant because it is expected to validate the TLR4/MD-2 complex as a novel target for improving the analgesic efficacy of opioids. The proposed research is innovative because it is the first drug discovery approach attempting to regulate opioid-induced glial activation while almost all previous research focused on neurons. The studies are built on a strong collaborative team with expertise that optimizes its chance to effectively bridge the atomic details of TLR4 activation with the macroscopic pain management inefficiencies of opioid use. Aim 1 and Aim 2 are two independent, parallel approaches that aim to attain highly specific inhibitors of the TLR4/MD-2 interactions. We aim to develop previously identified MD-2 peptides and T5342126 derivatives as potent, selective inhibitors of the TLR4/MD-2 complex with desired PK/PD properties. Aim 3 will test the working hypothesis that by disrupting the TLR4/MD- 2 complex, opioid-induced neuroinflammation can also be blocked, thus enhancing acute opioid analgesia. The optimized inhibitors from Aims 1 and 2 will be tested in vitro using various cell lines, in ex vivo microglia, as well as in animal models. The proposed studies, if successful, are projected to yield significant novel outcome: First, the results will further the understanding of the mechanism of clinically relevant opioid-induced neuroinflammation. Second, the TLR4/MD-2 complex will be validated as a feasible target for developing neuroinflammation suppressant. Third, the stapled peptide and small-molecule antagonists of the TLR4 pathway identified from the proposed research will serve as prototypes for potential drug candidates.